Sequence analysis of polymers has many practical applications. Of great interest is the ability to sequence the genomes of various organisms, including the human genome. Specific sequences can be recognized with a host of sequence-specific tagging methods such as various types of probes, engineered proteins, and also synthetic compounds. In any of these sequence-specific tagging approaches, there is always a need to resolve adjacent tags, in order to achieve higher resolution and thus map as much of the polymer as possible.
Linear analysis of DNA can be accomplished by analysis of fixed DNA molecules, analysis of moving DNA molecules, and analysis of DNA molecules using readers such as molecular motors or proteins capable of scanning along the length of a DNA strand. These approaches make use of a number of signals and detection systems to acquire the information from the sequence-specific tags on the polymer. For instance, fluorescence, atomic force microscopy (AFM), scanning tunneling microscopy (STM), as well as other electrical and electromagnetic methods, are suitable for capturing signals and thereby “reading” the sequence information of a polymer. All of these methods can be characterized and limited by their spatial resolution. Spatial resolution defines the minimum distance two adjacent probe molecules (e.g., sequence-specific tags) can be separated from each other and still be simultaneously detected as distinct, separate signals.
Fluorescence detection is often carried out by imaging. Optical resolution of fluorescence detection systems defines the smallest distance between probes at which they can still be distinguished. This distance is determined by diffraction. In a confocal microscopy system, in which the sample is illuminated and viewed through a pinhole in the image plane, the pinhole size determines the lateral resolution under uniform illumination of the pinhole. A confocal microscope system can be used in combination with a flow system that moves a target molecule (e.g., DNA or RNA) through a detection spot in the focal plane of the microscope. If the target molecules are stretched out in the direction of motion, and moved singly through the detection spot, then bound fluorescently labeled probes can be sequentially detected as they enter the detection spot. If the velocity of the target polymer is known, then the distance between detected probes can be determined from the time between sequential signals. According to prior art systems, probes that are spatially separated by more than the spot size can be distinguished from each other.
There is a need for increasing the resolution of detection systems in order to increase the amount of data captured from polymer analysis approaches.